Multimeric proteins are those composed of two or more polypeptide subunits, or promoters. A dimeric protein has two promoters, a tetramer has four promoters, and so on. In a particular multimeric protein, the component promoters associate in a specific way to produce the protein's native quaternary structure. For a dimer, such assembly or association is called dimerization. The promoters of a given multimeric protein may be identical to ("homo-") or different from ("hetero-") each other. For example, the homodimeric protein nerve growth factor (NGF) has two identical promoters. Promoters may differ in function within the multimeric protein. For example, the heterodimeric protein cholera toxin has a first subunit that permits penetration of the plasma membrane by the toxin and a second, non-identical subunit fit catalyzes the covalent modification of a cytosolic G-protein.
It is well known in biochemistry that the activity of a multimeric protein often depends on association of its component subunits. For example, the individual promoters of a particular enzyme may display no catalytic activity in isolation, but are only able to function when in a multimeric state. Similarly, various signal transduction pathways require ligand-induced self-association of cell surface receptors such as EGF-R, PDGF-R and the neurotrophin receptors for signalling to occur. Various ligands involved in signal transduction are themselves known to exist in multimeric form, e.g., NGF. There are several compelling reasons, from a neurological perspective, to be able to disrupt promoters involved in certain neurological processes.
The neurotrophins are a family of structurally and functionally related neurotrophic factors. The family includes prototypic member NGF, as well as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) (Heumann, Curr. Opin. Neurobiol. 4: 668-679 (1994)) and neurotrophin-6 (NT-6) (Gotz et at., Nature 372: 226-269 (1994)). The neurotrophins have similar structural conformations, including three surface .beta.-hairpin loops, a .beta.-strand, an internal reverse turn region, and N-and C-termini, and exhibit approximately 50% amino acid sequence identity.
Neurotrophins function to promote growth and survival of certain classes of peripheral and central neurons both during development and following neuronal damage. For example, NGF is involved in the development of neurons in the peripheral nervous system, supports neuronal survival, and enhances and maintains the differentiated state of neurons. Neurotrophins can promote neurite differentiation such as sprouting or process formation, and process growth. Neurotrophins can also modulate cell motility (Anton et al., Proc. Nat. Aced. Sci. USA 91: 2795-2799 (1994)), for example, both accelerate nerve process growth and decrease general cell motility. Another neurotrophin-mediated activity is induction of particular enzymes.
Furthermore, with respect to functional similarity, each of the neurotrophins can bind to a membrane bound receptor protein (MW.about.75 kDa) called the common neurotrophin receptor, or "p75.sup.NTR ". Each neurotrophin also binds with higher affinity to a second membrane-bound receptor protein of the tyrosine kinase receptor (Trk) family. In particular, NGF binds selectively to the TrkA receptor, and BDNF and NT-4/5 bind selectively to the TrkB receptor. NT-3 is less selective and, though it binds primarily to the TrkC receptor, NT3 also exhibits some binding to the TrkA and TrkB receptors (Ibanez et al., EMBO J. 12: 2281-2293 (1993)).
A variety of cell types express either p75.sup.NTR and/or a member of the Trk family of receptor tyrosine kinases. These include neurons, mast cells, glial cells such as astrocytes, oligodendrocytes and Schwann cells, and dysplasic or malignant cells such as neuroblastoma or melanoma cells. Cells of neuronal lineage that differentiate by extension of neurites in the presence of a neurotrophin in express both a member of the Trk receptor family and lower molecular weight receptor protein p75.sup.NTR.
NGF, a 118 amino acid protein, is an extremely important neurotrophin, being implicated in the pathogenesis of Alzheimer's disease, epilepsy and pain. The binding of NGF to its receptors is determined by distinct sequences within its primary amino acid structure The hairpin loop at residues 29-35 is responsible for recognition by p75.sup.NTR, while the amino and carboxy termini are important binding determinants for recognition by the TrkA receptor NGF exerts its biological activity as a non-covalent dimer. Two 118 residue NGF monomers are dimerized by hydrophobic and van der Waals interacts between their three anti-parallel pairs of .beta.-strands. Consequently, the amino terminus of a first NGF protomer and the carboxyl terminus of a second NGF protomer are spatially juxtaposed, and the amino terminus of the second NGF protomer and the carboxyl terminus of the first NGF protomer are spatially juxtaposed. Furthermore, although the NGF dimer thus has 2 pairs of termini, only one pair of termini is required for TrkA receptor recognition.
Variable domains within the NGF family of neurotrophins include the NGF NH2- and COOH-terminal residues (1-9 and 111-118, respectively) and certain internal residues that are revealed by the crystal structure of NGF (McDonald et al., Nature 354: 411-414 (1991)) to form four loop structures. These loops are three .beta. sheet structures, residues 25-35 (Loop 1, L1), 40-50 (Loop 2, L2) and 90-100 (Loop 4, L4); and a twisted loop (Loop 3, L3) residues 62-68.
It has been suggested previously that the variable regions mediate the biological effects of the neurotrophins, via specific Trk family receptors and the common neurotrophin receptor p75. This suggestion led to the employment of site-directed mutagenesis and recombinant chimeric protein techniques to demonstrate that specific residues within L2, 14 and the NH.sub.2 and COOH termini are required for Trk activation (Kahle et al., J. Biol Chem. 267: 22707-22710 (1992); Kullander et al., J. Neurosci. Res. 39: 195-210 (1994); Drinkwater et al., J. Biol. Chem. 268: 23202-23207 (1993)), and that domains of L1 and L4 are involved in p75 binding (Ibanez et al., EMBO J. 12: 2281-2293 (1993)). Monoclonal antibodies against antigenic determinants encompassing the NH.sub.2 and COOH termini and the L3 region of NGF implicate these domains in trkA receptor signalling (Nanduri et al., J. Neurosci. Res. 37: 433-444 (1994)).
Residues 58, 67, 68, 108, 109 and 110 are included in those residues that are conserved among the neurotrophins. The specific requirements of these residues with respect to NGF binding to receptors have not been examined using recombinant protein techniques, as they are required for protein structural integrity. The participation of NGF residues 60-67 in mediating interaction of NGF with either p75 or TrkA has been excluded by deletion mutagenesis studies (Drinkwater et al., J. Biol. Chem. 268: 23202-23207 (1993)).
Neurite growth is the best characterized differentiation response to NGF, and evidence is beginning to emerge that p75.sup.NTR can modulate this activity. Gene targeting studies resulting in nonfunctional p75.sup.NRT demonstrate reduced density of sensory and sympathetic innervation in vivo (Lee et al., Development 120: 1027-1033 (1994); Lee et al., Science 263: 1447-1449 (1994)), possibly related to a shift to the right of dose response curves for NGF (Davies et al., Neuron 11: 565-574 (1993)). Furthermore, evidence is beginning to emerge that p7em can activate apoptosis (Frade et al., Nature 166-168 (1996); Casaccia-Bonnefil et al. Nature 383: 716-719 (1996); Van der Zee et al., Science 274: 1729-1732 (1996)). Inhibiting p75.sup.NTR function may thus contribute to preventing of cell death.
The neurotrophins function primarily to promote survival of certain classes of peripheral and central neurons both during development and following neuronal damage. NGF, in particular, is involved with the development of neurons in the peripheral nervous system and supports neuronal survival, as well as enhancing and maintaining the differentiated state of neurons. Several lines of evidence also suggest that NGF may mediate inflammation (Levi-Montalcini, Science 237: 1154-1162 (1987)). However, in some neurological disease states, the neurotrophins may also support inappropriate neurite outgrowth thereby facilitating the progression of a disease condition. For example, neurotrophins promote the undesirable sprouting of hippocampal "mossy fibres". Such inappropriate sprouting of mossy fibers is a common accompaniment of epilepsy in humans. In other pathological states, such as Alzheimer's disease, as mentioned above, aberrant process growth, known as dystrophic neurite formation, is a strong correlate of disease severity.
Thus, although the neurotrophins are essential for the normal development and growth of neurons, they may be detrimental under certain circumstances. In such instances, factors capable of inhibiting or reducing selected neurotrophin-mediated activities, such as receptor binding and consequent signal transduction, would be desirable therapeutically to treat neurodegenerative disease and to repair nervous system injury.